Carbapenem-resistant Enterobacteriaceae (CRE), particularly those co-harboring multiple carbapenemase genes, pose a significant global health threat. However, the coexistence of bla (KPC-2) and bla (NDM-1) in Serratia sarumanii has not been previously reported. This study aims to report and characterize the first carbapenem-resistant S. sarumanii (CRSS) clinical strain MAS3954 in China co-harboring bla (KPC-2) and bla (NDM-1), focusing on its genetic characteristics, plasmid stability, and transfer potential. Whole-genome analysis revealed that the bla (KPC-2) and bla (NDM-1) were located on two distinct plasmids. Plasmid pMAS3954-KPC (113,856 bp, IncFII/IncFIB) exhibited low similarity (<66%) to known plasmids, indicating a novel fusion event between pKPC-h2 and S. marcescens chromosome, while pMAS3954-NDM (55,235 bp, IncX3) was highly conserved (100% identity/coverage). Conjugation experiments showed that the bla (NDM-1) was transferable, while bla (KPC-2) was not. During 10 days of continuous passage, the genetic context of bla (NDM-1) was gradually excised from the plasmids after the 8th day, whereas they maintained 100% retention for bla (KPC-2). S. sarumanii MAS3954 was multidrug-resistant (MDR), including carbapenems, β-lactams, β-lactam/β-lactamase inhibitors, trimethoprim-sulfamethoxazole, tetracycline, nitrofurantoin, colistin, and fluoroquinolones, but remained susceptible to certain aminoglycosides and tigecycline. Phylogenomic analysis identified a distinct clade for S. sarumanii MAS3954, diverging notably from other strains. Comparison of resistance genes further highlighted the unique co-harboring of bla (KPC-2) and bla (NDM-1) in MAS3954, absent in other strains. To our knowledge, this study represents the first characterization of clinical S. sarumanii strain co-harboring bla (KPC-2) and bla (NDM-1). The findings highlight the need for enhanced surveillance and infection control to prevent the spread of these MDR strains in healthcare settings.